Impact of primer sequence on TA cloning

Historically, I've had no difficulty with TOPO TA cloning, but I am currently having difficulty with lack of insert. Any number of approaches that I've taken recently have resulted in total absence of insert. I am aware that 5' bases can impact cloning efficiency (Peng et al 2007; Liu et al 2007) and wonder if that is the source of my problems. Below are details of my experiments and a list of the troubleshooting approaches we have taken.

Any comments are welcome!

Basic information: PCR from genomic DNA isolated via phenol/chloroform and ethanol precipitation (no proteinase K or RNAse digestion) ~6 primer sets against different targets, all with the same restriction sites on 5' termini; forward primer have a 5' NdeI site (CATATG); reverse primers have a 5' KpnI site (GGTACC)

Troubleshooting experiments and observations:(1) Control TOPO TA reactions with Invitrogen supplied pUC and primers yield =>90% cloning efficiency (direct colony PCR with T7/M13R) and many colonies(2) Control TOPO TA reactions work for 2 different TOPO kits (same competent cells)(3) Taq polymerase choice (NEB or Fermentas) makes no difference. Both should yield A-overhangs.(4) All primer sets yield the same difficulty.(5) Blue white selection - lots of blue colonies and a few (<0%) white colonies. White colonies do not include inserts.(6) Direct colony PCR of transformants with T7/M13R yields a small fragment - this may be the ~190 bp amplified polylinker but could include something else small.(7) Gel extraction didn't solve the problem, but # of transformants very low. Recircularization of the plasmid may have been the problem.(8) TOPO TA reactions of fresh PCR product and product stored several days in refrigerator yield equivalent results.

I am cloning a number of different targets (including RNAse H, superoxide dismutase, and a hypothetical protein) from trypanosomatid parasite DNA. It would surprise me were they all not compatible with cloning into E. coli, especially as I am not expressing them.

However, I have this difficulty with sequences that have previously been cloned in other labs with no difficulty.

I assume you have good, strong bands of the desired length in your PCR. I would clean up your pcr product with a column rather than a gel, eliminating primers and short fragments. I would make sure I had no band at 190 bp, even a weak one, which will have very high molarity since it is so short. You could amplify with a high fidelity enzyme (Phusion e.g.) then A tail with Taq and dATP. You might want to optimize your PCR. For trypanosomes, with very high AT content, you should lower the extension temperature to 64 degrees and lengthen the extension time by 1.5 - 2x. Given your trouble, I would pay attention to the Liu and Peng papers, but the A tailing with dATP should work.